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CN101674847A - Method for stabilizing a protein - Google Patents

Method for stabilizing a protein Download PDF

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CN101674847A
CN101674847A CN200880014420A CN200880014420A CN101674847A CN 101674847 A CN101674847 A CN 101674847A CN 200880014420 A CN200880014420 A CN 200880014420A CN 200880014420 A CN200880014420 A CN 200880014420A CN 101674847 A CN101674847 A CN 101674847A
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antibody
ser
protein solution
aqueous protein
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W·弗里斯
S·基泽
H-C·马勒
A·帕彭博格
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F Hoffmann La Roche AG
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61JCONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J1/00Containers specially adapted for medical or pharmaceutical purposes
    • A61J1/05Containers specially adapted for medical or pharmaceutical purposes for collecting, storing or administering blood, plasma or medical fluids ; Infusion or perfusion containers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591Stabilisation, fragmentation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61JCONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J3/00Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum

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Abstract

本发明涉及使水性蛋白溶液对抗外源胁迫而稳定的方法,该方法包括将所述溶液填装进容器以使所述容器在封闭时基本没有气体顶部空间的步骤。还提供了容器用于稳定水性蛋白溶液的用途。

Figure 200880014420

The present invention relates to a method of stabilizing an aqueous protein solution against exogenous stress comprising the step of filling said solution into a container such that said container is substantially free of gas headspace when closed. Also provided is the use of a container for stabilizing an aqueous protein solution.

Figure 200880014420

Description

稳定蛋白质的方法 Methods of Stabilizing Proteins

本发明涉及稳定水性蛋白溶液对抗外源胁迫的方法和稳定水性蛋白溶液的容器的用途。The present invention relates to a method for stabilizing an aqueous protein solution against exogenous stress and uses of a container for stabilizing the aqueous protein solution.

使水性蛋白溶液对抗外源胁迫(例如机械胁迫,例如振荡)而稳定仍然是制药工业的技术难题。已知下述事实:水性蛋白溶液在受到机械胁迫时具有聚集的趋向。在水性蛋白溶液(例如在容器中,例如药瓶中)运输期间几乎总会发生此类机械胁迫。Stabilizing aqueous protein solutions against exogenous stresses, such as mechanical stresses, such as shaking, remains a technical challenge for the pharmaceutical industry. It is known for the fact that aqueous protein solutions have a tendency to aggregate when subjected to mechanical stress. Such mechanical stress almost always occurs during transport of aqueous protein solutions (eg in containers, eg vials).

本领域中常用的解决方案是使用例如稳定剂如聚山梨酯进行物理-化学稳定,其中广泛使用聚山梨酯20(也称为Tween 20TM)或聚山梨酯80(Tween 80TM)、泊洛沙姆188或其它表面活性剂。A solution commonly used in the art is physico-chemical stabilization using for example stabilizers such as polysorbates, where polysorbate 20 (also known as Tween 20 ) or polysorbate 80 (Tween 80 ), porol Sham 188 or other surfactants.

但是,易于理解的是,不用化学产品进行稳定将有很多优点。具体而言,可能涉及到化学稳定剂对人体安全性的影响。因此,行政管理机构通常希望将药物制剂中包含的化学稳定剂的数量和含量都最小化。However, it is readily understood that stabilization without chemical products would have many advantages. Specifically, it may involve the impact of chemical stabilizers on human safety. Accordingly, regulatory agencies generally desire to minimize both the amount and amount of chemical stabilizers included in pharmaceutical formulations.

与健康问题相比的次要考虑是为了稳定水性蛋白溶液必须加入的化学稳定剂的成本。A secondary consideration compared to health concerns is the cost of the chemical stabilizers that must be added to stabilize the aqueous protein solution.

因此,很清楚的是,需要不使用化学稳定剂来使水性蛋白溶液对抗外源胁迫而稳定的方法。Therefore, it is clear that there is a need for methods of stabilizing aqueous protein solutions against exogenous stresses without the use of chemical stabilizers.

对于该目的,本申请人发现,使用包括将所述水性蛋白溶液填装入容器中,以使得容器在封闭时基本没有气体顶部空间的步骤的方法,令人吃惊地能够在不使用稳定剂的条件下使水性蛋白溶液对抗外源胁迫(例如机械胁迫)而稳定。For this purpose, the applicants have found that using a method comprising the step of filling said aqueous protein solution into a container such that the container is substantially free of gas headspace when closed, surprisingly enables The conditions stabilize the aqueous protein solution against exogenous stress, such as mechanical stress.

考虑到克服了通常存在的技术偏见,该发现甚至更加令人吃惊。事实上,本发明的方法克服了公知的技术偏见,即必须使用化学稳定剂来使水性蛋白溶液对抗外源胁迫而得到稳定。The finding is even more surprising given that usual technical biases were overcome. In fact, the method of the present invention overcomes the well-known technical prejudice that chemical stabilizers must be used to stabilize aqueous protein solutions against exogenous stresses.

图1显示了根据本发明的方法可在水溶液中被稳定的Aβ抗体的糖基化位点。Figure 1 shows the glycosylation sites of the Aβ antibody that can be stabilized in aqueous solution according to the method of the present invention.

图2显示了10mg/ml抗体(Mab A)制剂于5℃在振荡胁迫下在6ml瓶中随时间进程的情况,其中使用3种填装体积(A)2.5ml(B)5.3ml(C)9.0ml,并且有不同的PS20的量(■0%;□0.0025%;▲0.005%;△0.01%),这是通过可见的颗粒计数、浊度和可溶的聚集产物来分析的。Figure 2 shows the time course of a 10mg/ml antibody (Mab A) formulation in a 6ml bottle under shaking stress at 5°C using 3 fill volumes (A) 2.5ml (B) 5.3ml (C) 9.0 ml with varying amounts of PS20 (■ 0%; □ 0.0025%; ▲ 0.005%; Δ0.01%), which were analyzed by visible particle counts, turbidity and soluble aggregation products.

图3显示了10mg/ml抗体(Mab A)制剂于25℃在振荡胁迫下在6ml瓶中随时间进程的情况,其中使用3种填装体积(A)2.5ml(B)5.3ml(C)9.0ml,并且有不同的PS20的量(■0%;□0.0025%;▲0.005%;△0.01%),这是通过可见的颗粒计数、浊度和可溶的聚集产物来分析的。Figure 3 shows the time course of a 10mg/ml antibody (Mab A) formulation in a 6ml bottle under shaking stress at 25°C using 3 fill volumes (A) 2.5ml (B) 5.3ml (C) 9.0 ml with varying amounts of PS20 (■ 0%; □ 0.0025%; ▲ 0.005%; Δ0.01%), which were analyzed by visible particle counts, turbidity and soluble aggregation products.

图4显示了10mg/ml抗体(Mab A)制剂于5℃在振荡胁迫下在6ml瓶中随时间进程的情况,其中使用3种填装体积(A)2.5ml(B)5.3ml(C)9.0ml,并且有不同的PS20的量(■0%;□0.0025%;▲0.005%;△0.01%),这是通过对每ml中≥2μm、≥10μm和≥25μm的镜检可见的(sub-visible)颗粒来分析的。Figure 4 shows the time course of a 10 mg/ml antibody (Mab A) formulation at 5°C under shaking stress in a 6ml vial using 3 fill volumes (A) 2.5ml (B) 5.3ml (C) 9.0ml, and there are different amounts of PS20 (0%; 0.0025%; ▲0.005%; 0.01%), which is visible by microscopic examination of ≥2 μm, ≥10 μm and ≥25 μm in each ml (sub -visible) particles are analyzed.

图5显示了10mg/ml抗体(Mab A)制剂于25℃在振荡胁迫下在6ml瓶中随时间进程的情况,其中使用3种填装体积(A)2.5ml(B)5.3ml(C)9.0ml,并且有不同的PS20的量(■0%;□0.0025%;▲ 0.005%;△0.01%),这是通过对每ml中≥2μm、≥10μm和≥25μm的镜检可见的颗粒来分析的。Figure 5 shows the time course of a 10 mg/ml antibody (Mab A) formulation at 25°C under shaking stress in a 6ml bottle using 3 fill volumes (A) 2.5ml (B) 5.3ml (C) 9.0ml, and there are different amounts of PS20 (0%; 0.0025%; 0.005%; 0.01%), which is determined by microscopically visible particles of ≥2 μm, ≥10 μm and ≥25 μm per ml analysis.

图6显示了两种10mg/ml的抗体Mab A■和Mab B

Figure A20088001442000051
在(A)非胁迫条件下和25℃振荡胁迫72小时条件下的聚集物形成,其在6ml瓶中使用3种填装体积(B)2.5ml(C)5.3ml(D)9.0ml,并且有不同的PS20的量(0%;0.0025%;0.005%),这是通过可见的颗粒计数、浊度和可溶的聚集产物来分析的。Figure 6 shows two 10mg/ml antibodies Mab A and Mab B
Figure A20088001442000051
Aggregate formation under (A) non-stress conditions and 25°C shaking stress for 72 hours using 3 fill volumes in 6ml bottles (B) 2.5ml (C) 5.3ml (D) 9.0ml, and There were varying amounts of PS20 (0%; 0.0025%; 0.005%), which were analyzed by visible particle counts, turbidity and soluble aggregation products.

图7显示了两种10mg/ml的抗体Mab A■和Mab B

Figure A20088001442000052
在(A)非胁迫条件下和25℃振荡胁迫72小时条件下的聚集物形成,其在6ml瓶中使用3种填装体积(B)2.5ml(C)5.3ml(D)9.0ml,并且有不同的PS20的量(0%;0.0025%;0.005%),这是通过对每ml中≥2μm、≥10μm和≥25μm的镜检可见的颗粒来分析的。Figure 7 shows two 10mg/ml antibodies Mab A and Mab B
Figure A20088001442000052
Aggregate formation under (A) non-stress conditions and 25°C shaking stress for 72 hours using 3 fill volumes in 6ml bottles (B) 2.5ml (C) 5.3ml (D) 9.0ml, and There were different amounts of PS20 (0%; 0.0025%; 0.005%), which were analyzed by microscopically visible particles >2 μm, >10 μm and >25 μm per ml.

图8和9显示了外源胁迫(此处是机械胁迫)之后填装有水性蛋白溶液的容器(此处是可药用的瓶)的照片。左侧显示了与现有技术类似的在封闭时具有气体顶部空间的含有蛋白溶液的容器。右侧显示了根据本发明方法的在封闭时基本没有气体顶部空间的含有蛋白溶液的容器。Figures 8 and 9 show photographs of containers (here pharmaceutically acceptable bottles) filled with aqueous protein solutions after exogenous stress (here mechanical stress). The left shows a container containing a protein solution with a gas headspace when closed similar to the prior art. The right side shows a container containing a protein solution that is substantially free of gas headspace when closed according to the method of the invention.

术语“稳定”是指:用下文实施例中描述的相应试验进行测量时,水性蛋白溶液基本上不含显著量的聚集物和/或浑浊和/或镜检可见的颗粒和/或可见的颗粒。The term "stable" means that the aqueous protein solution is substantially free of significant amounts of aggregates and/or cloudiness and/or microscopic particles and/or visible particles as measured by the corresponding test described in the Examples below .

“外源胁迫”这种表述指通过外源作用诱导的对水性蛋白溶液的胁迫。外源作用指来自包含容器和水性蛋白溶液的系统之外的作用。外源胁迫的一种例子是机械胁迫。机械胁迫的一种例子是振荡。在制药工业中的振荡可偶然发生(例如在运输期间)或主动发生(例如用于匀化溶液)。The expression "exogenous stress" refers to stress induced by exogenous action on an aqueous protein solution. Exogenous effects refer to effects from outside the system comprising the container and the aqueous protein solution. An example of exogenous stress is mechanical stress. An example of mechanical stress is oscillation. Shaking in the pharmaceutical industry can occur accidentally (eg during transport) or actively (eg for homogenizing solutions).

术语“容器”指可药用容器。合适的容器包含与开关装置相连的容纳器(recipient)部分。优选地,容器由与开关装置相连的容纳器构成,例如常规的带有封闭盖子的可药用瓶、预装填的注射器、胶囊或安瓿。进一步更优选地,容器是带有封闭盖子的常规可药用瓶。容器可装有密封关闭装置,例如,对瓶起密封关闭作用的盖子,防止周围外界大气进入水性蛋白溶液。The term "container" refers to a pharmaceutically acceptable container. A suitable container comprises a recipient portion connected to the switching device. Preferably, the container consists of a receptacle, such as a conventional pharmaceutical bottle with a closed cap, a prefilled syringe, capsule or ampoule, connected to the closure means. Still more preferably, the container is a conventional pharmaceutically acceptable bottle with a closed cap. The container may be provided with a hermetic closure, eg, a lid that provides a hermetic closure to the bottle, preventing the ambient atmosphere from entering the aqueous protein solution.

“水性蛋白溶液”这种表述指含有蛋白的水性溶液。蛋白质浓度可以是0.01-280mg/mL。The expression "aqueous protein solution" refers to an aqueous solution containing protein. The protein concentration can be 0.01-280 mg/mL.

“在封闭时基本没有气体顶部空间”这种表述表示:用水性蛋白溶液填装容器的本领域技术人员将容器填装至该容器的最大体积,所述最大体积是通过例如目测或者通过计算预先确定的。这可通过例如用水性蛋白溶液将容器填装至容器的最大可能体积来实现,测量的精密度通过对所述溶液在最大体积时形成的凹液面在实验室目测确定,或通过对容器称重而从重量上测定,或者使用本领域已知的标准设备通过体积测定,其通常是制药工业在规模生产时的所用方法。填装体积表示仍旧足以使容器关闭而不会溢出。The expression "substantially free of gas headspace when closed" means that a person skilled in the art who fills a container with an aqueous protein solution fills the container to the maximum volume of the container predetermined by, for example, visual inspection or by calculation. definite. This can be achieved, for example, by filling the container with an aqueous protein solution to the maximum possible volume of the container, the precision of the measurement being determined in the laboratory by visual inspection of the meniscus formed by said solution at its maximum volume, or by weighing the container. Weight, measured gravimetrically, or by volume using standard equipment known in the art, is generally the method used by the pharmaceutical industry on scale. The fill volume indicated is still sufficient to close the container without spilling.

因此,术语“基本”在与“在封闭时基本没有气体顶部空间”这种表述一起使用时表示目测的、采用重量或体积测定的最大体积和/或没有气体顶部空间的精确度,其取决于目测时操作填装者的精确度或者在采用重量或体积测定时所用设备的精确度。Thus, the term "substantially" when used in conjunction with the expression "substantially free of gas headspace when enclosed" means the accuracy of visual, gravimetric or volumetric maximum volume and/or absence of gas headspace, depending on The accuracy of the operator operating the filler when visually determined or the accuracy of the equipment used when gravimetric or volumetric determinations are used.

术语“通常可药用的赋形剂”是指施用给人类或动物的市售产品或其它研发产品中已经含有的赋形剂。该术语包含下列类别的赋形剂:缓冲剂(包括柠檬酸盐、乙酸盐、琥珀酸盐、磷酸盐、组氨酸、甘氨酸、精氨酸缓冲液)、氨基酸(包括精氨酸、甘氨酸、赖氨酸、色氨酸、甲硫氨酸)、糖或糖醇(包括蔗糖、海藻糖、甘露醇、山梨醇)、表面活性剂(包括聚山梨酯20、聚山梨酯80、泊洛沙姆188、十二烷基硫酸钠、Triton X)和其它赋形剂(例如聚乙烯吡咯烷酮、环糊精、聚乙二醇),等等。The term "generally pharmaceutically acceptable excipients" refers to excipients already contained in marketed products or other products under development for administration to humans or animals. The term includes the following classes of excipients: buffers (including citrate, acetate, succinate, phosphate, histidine, glycine, arginine buffers), amino acids (including arginine, glycine , lysine, tryptophan, methionine), sugars or sugar alcohols (including sucrose, trehalose, mannitol, sorbitol), surfactants (including polysorbate 20, polysorbate 80, porol Sham 188, sodium lauryl sulfate, Triton X) and other excipients (such as polyvinylpyrrolidone, cyclodextrin, polyethylene glycol), and the like.

术语“单克隆抗体”或“单克隆抗体组合物”在本文中使用时指单种氨基酸组成的抗体分子的制备物。因此,术语“人单克隆抗体”指显示出单种结合特异性的抗体,其具有源于人类种系的免疫球蛋白序列的可变区和恒定区。在一种实施方式中,人单克隆抗体通过杂交瘤产生,所述杂交瘤包括与永生化细胞融合的B细胞,该B细胞来自于转基因的非人动物(例如转基因小鼠),具有包含人重链转基因和人轻链转基因的基因组。The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules composed of a single amino acid. Thus, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells fused with immortalized cells from a transgenic non-human animal (eg, a transgenic mouse) with human Genomes of heavy chain transgene and human light chain transgene.

抗体分子作为蛋白质药物类的一部分,非常易于物理和化学降解,例如变性和聚集、脱酰胺、氧化和水解。蛋白质稳定性受蛋白自身特性(例如氨基酸序列)影响和外界影响(例如温度、溶剂pH、赋形剂、界面、振荡或剪切率)的影响。因此,确定最佳的制剂条件以保护蛋白质在生产、贮藏和给药过程中避免降解反应是很重要的。Antibody molecules, as part of protein drug classes, are highly susceptible to physical and chemical degradation such as denaturation and aggregation, deamidation, oxidation and hydrolysis. Protein stability is affected by the properties of the protein itself (such as amino acid sequence) and external influences (such as temperature, solvent pH, excipients, interfaces, oscillation or shear rate). Therefore, it is important to determine the optimal formulation conditions to protect proteins from degradation reactions during production, storage and administration.

术语“抗IGF-1R人单克隆抗体”或“huMAb IGF-IR”是指在WO2005/005635中所述的和要求保护的抗体,该文献的内容、尤其是权利要求引入本文作为参考。The term "anti-IGF-1R human monoclonal antibody" or "huMAb IGF-IR" refers to the antibody described and claimed in WO2005/005635, the contents of which, especially the claims, are incorporated herein by reference.

“Aβ”指能够特异性结合淀粉样蛋白-β肽的Aβ抗体(或其混合物)。特异性结合Aβ的抗体是本领域已知的。能用于本发明的制剂中的Aβ抗体的具体例子已在公开的PCT专利申请WO 03/070760中、尤其是权利要求中被描述过,该文献的内容通过引用并入本文。"A[beta]" refers to an A[beta] antibody (or mixture thereof) capable of specifically binding amyloid-[beta] peptide. Antibodies that specifically bind A[beta] are known in the art. Specific examples of A[beta] antibodies that can be used in the formulations of the invention are described, especially in the claims, in published PCT patent application WO 03/070760, the contents of which are incorporated herein by reference.

淀粉样蛋白-β肽也被称为“β淀粉样蛋白”、“Aβ”、“Aβ4”或“β-A4”,特别是在本发明的上下文中被称为“Aβ”,是与形成淀粉样病变的疾病例如阿尔茨海默症有关的细胞外神经斑的主要组分;参见,Selkoe(1994),Ann.Rev.Cell Biol.10,373-403,Koo(1999),PNAS Vol.96,pp.9989-9990,US4,666,829或Glenner(1984),BBRC 12,1131。该β淀粉样蛋白源于“阿尔茨海默前体蛋白/β-淀粉样蛋白前体蛋白”(APP)。APP是整合膜糖蛋白(见Sisodia(1992),PNAS Vol.89,pp.6075),其内的Aβ序列被一种质膜蛋白酶α-分泌酶进行内源性蛋白酶切除(见Sisodia(1992),如上述文献)。而且,其它一些分泌酶活性,特别是β-分泌酶和γ-分泌酶活性导致了包含39个氨基酸(Aβ39)、40个氨基酸(Aβ40)、42个氨基酸(Aβ42)或43个氨基酸(Aβ43)的β淀粉样蛋白(Aβ)的细胞外释放;见Sinha(1999),PNAS 96,11094-1053;Price(1998),Science 282,1078-1083;WO 00/72880或Hardy(1997),TINS20,154。Amyloid-beta peptide, also known as "beta-amyloid", "Aβ", "Aβ4" or "beta-A4", especially in the context of the present invention as "Aβ", is associated with the formation of amyloid major component of extracellular plaques associated with diseases such as Alzheimer's disease; see, Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo (1999), PNAS Vol.96 , pp.9989-9990, US 4,666,829 or Glenner (1984), BBRC 12, 1131. This β-amyloid is derived from "Alzheimer Precursor Protein/β-Amyloid Precursor Protein" (APP). APP is an integral membrane glycoprotein (see Sisodia (1992), PNAS Vol.89, pp.6075) in which the Aβ sequence is cleaved by endogenous proteases by a plasma membrane protease α-secretase (see Sisodia (1992) , as mentioned above). Moreover, some other secretase activities, particularly β-secretase and γ-secretase activities, lead to β-secretases containing 39 amino acids (Aβ39), 40 amino acids (Aβ40), 42 amino acids (Aβ42) or 43 amino acids (Aβ43) Extracellular release of β-amyloid (Aβ); see Sinha (1999), PNAS 96, 11094-1053; Price (1998), Science 282, 1078-1083; WO 00/72880 or Hardy (1997), TINS20, 154.

Aβ具有几种天然存在的形式,其中人的形式被称为上述的Aβ39、Aβ40、Aβ41、Aβ42和Aβ43。最主要的形式Aβ42具有下述氨基酸序列(从N-末端开始):A[beta] has several naturally occurring forms, of which the human forms are known as A[beta]39, A[beta]40, A[beta]41, A[beta]42 and A[beta]43 as described above. The most dominant form, Aβ42, has the following amino acid sequence (starting from the N-terminus):

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO:3)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3)

在Aβ41、Aβ40、Aβ39中,分别丢失了C-末端的氨基酸A、IA和VIA。在Aβ43形式中,在上述序列(SEQ ID NO:3)的C-末端包含额外的苏氨酸残基。In Aβ41, Aβ40, Aβ39, the C-terminal amino acids A, IA and VIA are missing, respectively. In the Aβ43 form, an additional threonine residue is included at the C-terminus of the above sequence (SEQ ID NO: 3).

合适的Aβ抗体是免疫球蛋白分子,例如IgG分子。IgG特征在于包含两条重链和两条轻链(如图1所示),这些分子包含两个抗原结合位点。所述抗原结合位点包含由重链的部分(VH)和轻链的部分(VL)构成的“可变区”。抗原结合位点通过VH和VL结构域并列形成。关于抗体分子或免疫球蛋白分子的一般信息,还可参见常用教科书,例如Abbas“Cellular andMolecular Immunology”,W.B.Sounders Company(2003)。Suitable A[beta] antibodies are immunoglobulin molecules, such as IgG molecules. IgG is characterized by comprising two heavy chains and two light chains (as shown in Figure 1), and these molecules contain two antigen binding sites. The antigen binding site comprises a "variable region" consisting of part of the heavy chain (VH) and part of the light chain (VL). The antigen binding site is formed by the juxtaposition of VH and VL domains. General information on antibody molecules or immunoglobulin molecules can also be found in commonly used textbooks, eg Abbas "Cellular and Molecular Immunology", W.B. Sounders Company (2003).

在一种实施方式中,本发明的水性蛋白溶液中存在的蛋白是Aβ抗体(或此类抗体的混合物),其中所述抗体的重链中至少一个可变区包含N-糖基化。重链的可变区(VH)中糖基化的天冬酰胺(Asn)可以处于互补性决定区域2(CDR2区域)中,所述糖基化的天冬酰胺(Asn)可处于SEQ ID NO:1所示的重链可变区(VH)的第52位上。In one embodiment, the protein present in the aqueous protein solution of the invention is an Aβ antibody (or a mixture of such antibodies), wherein at least one variable region in the heavy chain of the antibody comprises N-glycosylation. The glycosylated asparagine (Asn) in the variable region (VH) of the heavy chain can be in the complementarity determining region 2 (CDR2 region), and the glycosylated asparagine (Asn) can be in SEQ ID NO : 1 at position 52 of the heavy chain variable region (VH).

术语“单糖基化抗体”指在单个抗体分子的一个(VH)区域中包含N-糖基化的抗体分子;也见图1。术语“双糖基化抗体”定义了在重链的两个可变区都被N-糖基化的抗体分子(图1)。在两个重链(VH)结构域上都没有N-糖基化的抗体分子被称为“非糖基化抗体”(图1)。单糖基化抗体、双糖基化抗体和非糖基化抗体可包含相同的氨基酸序列或不同的氨基酸序列。The term "monoglycosylated antibody" refers to an antibody molecule comprising N-glycosylation in one ( VH ) region of a single antibody molecule; see also Figure 1 . The term "double glycosylated antibody" defines an antibody molecule that is N-glycosylated in both variable regions of the heavy chain (Figure 1). Antibody molecules that lack N-glycosylation on both heavy chain ( VH ) domains are referred to as "aglycosylated antibodies" (Figure 1). Monoglycosylated antibodies, double glycosylated antibodies, and aglycosylated antibodies can comprise the same amino acid sequence or different amino acid sequences.

单糖基化抗体和双糖基化抗体在本文中被称为“糖基化抗体亚型”。经纯化的抗体分子其特征为至少一个抗原结合位点包含在重链可变区(VH)中的糖基化,是指单糖基化抗体,其不含选自双糖基化抗体和非糖基化抗体的亚型或者仅含有非常低的量,即“经纯化的单糖基化抗体”。本发明内容中的双糖基化抗体不含选自单糖基化抗体和非糖基化抗体的亚型或仅含有非常低的量,即“经纯化的双糖基化抗体”。Monoglycosylated antibodies and double glycosylated antibodies are referred to herein as "glycosylated antibody subtypes". A purified antibody molecule characterized by at least one antigen-binding site comprising glycosylation in the heavy chain variable region (VH) refers to a monoglycosylated antibody that is free of antibodies selected from the group consisting of double glycosylated antibodies and non-glycosylated antibodies. Subtypes of glycosylated antibodies are either contained only in very low amounts, ie "purified monoglycosylated antibodies". The double glycosylated antibody in the context of the present invention does not contain a subtype selected from monoglycosylated antibodies and aglycosylated antibodies or only contains a very low amount, ie "purified double glycosylated antibody".

根据本发明方法的水性蛋白溶液中的蛋白质可以是单糖基化或双糖基化或非糖基化的抗体,或其被具体定义的混合物。本文提供的抗体混合物或抗体库可以包含50%的如本文所述的单糖基化抗体和50%的双糖基化抗体。但是,30/70至70/30的比例也是本发明所包括的。并且,本领域技术人员知道,本发明的抗体混合物也包括其它比例。例如,10/90或90/10、20/80或80/20以及40/60或60/40的比例也可用于本发明的范围。本发明的方法中所包括的抗体混合物包含如上文所定义的双糖基化和单糖基化抗体,其特别有用的比例是40/60至45/55。The protein in the aqueous protein solution according to the method of the invention may be a monoglycosylated or diglycosylated or aglycosylated antibody, or a specifically defined mixture thereof. The antibody mixture or antibody library provided herein can comprise 50% monoglycosylated antibodies as described herein and 50% diglycosylated antibodies. However, ratios of 30/70 to 70/30 are also encompassed by the present invention. Moreover, those skilled in the art know that the antibody mixture of the present invention also includes other ratios. For example, ratios of 10/90 or 90/10, 20/80 or 80/20 and 40/60 or 60/40 are also within the scope of the present invention. The antibody mixture comprised in the method of the invention comprises double and monoglycosylated antibodies as defined above in a particularly useful ratio of 40/60 to 45/55.

术语“不含或仅含有非常低的量”是指完全不存在各种其它(糖基化)亚型,或其它(糖基化)亚型以至多10%,例如至多5%,例如至多4%,例如至多3%,例如至多2%,例如至多1%,例如至多0.5%,例如至多0.3%,例如至多0.2%的浓度存在。The term "free or only in very low amounts" means that each other (glycosylated) isoform is completely absent, or that other (glycosylated) isoforms are up to 10%, such as up to 5%, such as up to 4% %, such as at most 3%, such as at most 2%, such as at most 1%, such as at most 0.5%, such as at most 0.3%, such as at most 0.2%.

术语“抗体”在本文中使用时与术语“抗体分子”同义,其在本发明的范畴中包括抗体分子,例如完整的免疫球蛋白分子,例如IgMs、IgDs、IgEs、IgAs或IgGs(例如IgG1、IgG2、IgG2b、IgG3或IgG4)以及此类免疫球蛋白分子的部分,例如Fab片段、Fab’片段、F(ab)2片段、嵌合F(ab)2或嵌合Fab’片段、嵌合Fab片段或经分离的VH区域或CDR区域(所述经分离的VH区域或CDR区域是例如被整合或加工在相应的“框架”中)。因此,术语“抗体”还包含免疫球蛋白的已知亚型和修饰物,例如单链抗体或单链Fv片段(scAB/scFv)或双特异性抗体构建体,所述亚型和修饰物的特征在于包含至少一个本文所定义的糖基化的VH区域。此类亚型或修饰物的一个具体例子可以是VH-VL或VL-VH形式的sc(单链)抗体,其中所述VH包含本文所述的糖基化。双特异性的scFv也包括在本文内,例如VH-VL-VH-VL、VL-VH-VH-VL、VH-VL-VL-VH形式。术语“抗体”还包括双抗体和包含与至少一个抗原结合部位/肽连接的作为载体的抗体Fc结构域的分子,例如WO 00/24782所述的肽抗体。从上文明显可知,用于本发明方法中的水性蛋白溶液还包括含有抗体/抗体分子的混合物的水性Aβ抗体溶液。所述抗体的一种具体“混合物”是上文所述的,即,抗Aβ的“单”和“双”糖基化抗体的混合物。The term "antibody" as used herein is synonymous with the term "antibody molecule", which includes within the scope of the invention antibody molecules such as intact immunoglobulin molecules such as IgMs, IgDs, IgEs, IgAs or IgGs (such as IgG1 , IgG2, IgG2b, IgG3 or IgG4) and portions of such immunoglobulin molecules such as Fab fragments, Fab' fragments, F(ab)2 fragments, chimeric F(ab)2 or chimeric Fab' fragments, chimeric Fab fragments or isolated VH regions or CDR regions eg integrated or processed in a corresponding "framework"). Thus, the term "antibody" also encompasses known subtypes and modifications of immunoglobulins, such as single chain antibodies or single chain Fv fragments (scAB/scFv) or bispecific antibody constructs, of which subtypes and modifications A VH region characterized by comprising at least one glycosylation as defined herein. A specific example of such a subtype or modification may be a sc (single chain) antibody in the VH-VL or VL-VH format, wherein the VH comprises a glycosylation as described herein. Also included herein are bispecific scFvs, eg, VH-VL-VH-VL, VL-VH-VH-VL, VH-VL-VL-VH formats. The term "antibody" also includes diabodies and molecules comprising the Fc domain of an antibody as a carrier linked to at least one antigen binding site/peptide, such as peptibodies as described in WO 00/24782. As evident from the above, the aqueous protein solution used in the method of the present invention also includes an aqueous Aβ antibody solution containing a mixture of antibodies/antibody molecules. One particular "mixture" of said antibodies is that described above, ie a mixture of "mono" and "double" glycosylated antibodies against A[beta].

“抗体片段”还包括本身不能提供效应物功能(ADCC/CDC),但在与合适的抗体恒定结构域组合后能以本发明的方式提供该功能的此类片段。"Antibody fragment" also includes such fragments which do not provide effector function (ADCC/CDC) by themselves, but are capable of providing this function in the manner of the invention when combined with a suitable antibody constant domain.

Aβ抗体可用作为本发明方法中的水性蛋白溶液中的蛋白质,其包括重组产生的Aβ抗体。这些抗体可在哺乳动物细胞培养系统,例如CHO细胞中产生。此类哺乳动物细胞培养系统特别是用于制备Aβ抗体或糖基化的Aβ抗体/抗体分子,如本文具体例举的在可变区包含N-糖基化的Aβ抗体。抗体分子还可通过系列的色谱或过滤步骤来纯化,例如按照下文所述纯化特异性的糖基化抗体亚型。Antibodies to A[beta] can be used as proteins in the aqueous protein solution in the methods of the invention, including recombinantly produced antibodies to A[beta]. These antibodies can be produced in mammalian cell culture systems, such as CHO cells. Such mammalian cell culture systems are particularly useful for the production of A[beta] antibodies or glycosylated A[beta] antibodies/antibody molecules, such as the A[beta] antibodies comprising N-glycosylation in the variable region as specifically exemplified herein. Antibody molecules may also be purified by a series of chromatographic or filtration steps, eg, for the purification of specific glycosylated antibody isoforms as described below.

术语“单克隆抗体”或“单克隆抗体组合物”在本文中使用时指单种氨基酸组成的抗体分子的制备物。因此,术语“人单克隆抗体”指显示出单种结合特异性的抗体,其具有源于人类种系的免疫球蛋白序列的可变区和恒定区。在一种实施方式中,人单克隆抗体通过杂交瘤产生,所述杂交瘤包括与永生化细胞融合的B细胞,该B细胞来自于转基因的非人动物(例如转基因小鼠),具有包含人重链转基因和人轻链转基因的基因组。The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules composed of a single amino acid. Thus, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells fused with immortalized cells from a transgenic non-human animal (eg, a transgenic mouse) with human Genomes of heavy chain transgene and human light chain transgene.

Aβ抗体可包含或具有如SEQ ID NO:1定义的可变区:The Aβ antibody may comprise or have a variable region as defined by SEQ ID NO: 1:

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

该序列也在下文中阐述,其中指出了CDR、CH区域、重区域以及两个N-糖基化位点(Asn52和Asn306):The sequence is also set forth below, where the CDRs, CH region, heavy region, and two N-glycosylation sites (Asn52 and Asn306) are indicated:

QVELVESGGGLVQPGGSLRLSCAASWVRQAPGKGLEWVS

Figure A20088001442000112
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
Figure A20088001442000113
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCP
Figure A20088001442000114
Figure A20088001442000116
Figure A20088001442000117
Figure A20088001442000118
Figure A20088001442000119
(SEQ ID NO:1)QVELVESGGGLVQPGGSLRLSCAAS WVRQAPGKGLEWVS
Figure A20088001442000112
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
Figure A20088001442000113
WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKV EPKSCDKTHTCP
Figure A20088001442000114
Figure A20088001442000116
Figure A20088001442000117
Figure A20088001442000118
Figure A20088001442000119
(SEQ ID NO: 1)

Figure A200880014420001110
:CDR1、2、3
Figure A200880014420001110
: CDR1, 2, 3

下划线:CH1 Underscore : CH1

Figure A200880014420001111
:铰链
Figure A200880014420001111
:Hinge

Figure A200880014420001112
:CH2
Figure A200880014420001112
: CH2

Figure A200880014420001113
:CH3
Figure A200880014420001113
: CH3

粗体N:N-连接的糖基化位点Bold N: N-linked glycosylation site

本文所述的包含SEQ ID NO:1的示例性Aβ抗体还可包含轻链,所述轻链可包含或具有下述氨基酸序列:Exemplary Aβ antibodies described herein comprising SEQ ID NO: 1 may also comprise a light chain which may comprise or have the following amino acid sequence:

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2)DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2)

术语“Aβ抗体A”在本文中使用时指包含如SEQ ID NO:1所定义的重链和如SEQ ID NO:2所定义的轻链的Aβ抗体。The term "Aβ antibody A" as used herein refers to an Aβ antibody comprising a heavy chain as defined in SEQ ID NO: 1 and a light chain as defined in SEQ ID NO: 2.

术语“单糖基化抗体”在本文中使用时指在单个抗体分子(例如免疫球蛋白,例如IgG,例如IgG1)的一个(VH)区域中包含N-糖基化的抗体分子。例如,所述“单糖基化形式”包含在重链的一个可变区上的糖基化,例如在本文所述的“Aβ抗体A”的天冬酰胺位置“Asn52”上。如本文所述,该“单糖基化的IgG1形式或单糖基化的亚型”还可包括在Fc部分的高度保守的糖基化位点中的糖基化,例如在本文示例的“Aβ抗体A”的非可变Fc部分中的天冬酰胺Asn306。The term "monoglycosylated antibody" as used herein refers to an antibody molecule comprising N-glycosylation in one (VH) region of a single antibody molecule (eg immunoglobulin, eg IgG, eg IgGl). For example, said "monoglycosylated form" comprises glycosylation on one variable region of the heavy chain, for example at the asparagine position "Asn52" of "Aβ antibody A" described herein. As described herein, this "monoglycosylated IgG1 form or monoglycosylated subtype" may also include glycosylation in highly conserved glycosylation sites of the Fc portion, such as exemplified herein in " Asparagine Asn306 in the non-variable Fc part of Aβ antibody A".

在本发明的含义中,术语“双糖基化抗体”包含如本文定义的在重链(VH)区域的两个可变区上的糖基化。该“双糖基化形式”还包含在两条重链的可变区上的糖基化,例如,在本文所述的“Aβ抗体A”的天冬酰胺位置“Asn52”上。该“双糖基化的IgG1形式或双糖基化的亚型”还可包括如本文所述在非可变/恒定的Fc部分中高度保守的糖基化位点上的糖基化,特别是在示例的“Aβ抗体A”的第306位上。所附的图1说明了相应的抗体分子。Within the meaning of the present invention, the term "double glycosylated antibody" comprises glycosylation as defined herein on both variable regions of the heavy chain (VH) region. This "biglycosylated form" also comprises glycosylation on the variable regions of both heavy chains, for example, at the asparagine position "Asn52" of "Aβ antibody A" described herein. This "double-glycosylated IgG1 form or double-glycosylated subtype" may also include glycosylation at highly conserved glycosylation sites in the non-variable/constant Fc portion as described herein, in particular It is at position 306 of the exemplary "Aβ antibody A". The accompanying Figure 1 illustrates the corresponding antibody molecule.

在可变区(例如重链的两个可变区(两个(VH)区域))没有此类翻译后修饰的抗体在本发明范畴内被视为“非糖基化形式”,包括在重链的可变区没有糖基化的。但是,该“非糖基化形式”可在抗体的恒定区(C-区域)包含糖基化,例如,最常见是在Fc部分的高度保守的糖基化位点上,特别是本文所定义的非可变/恒定的Fc部分的天冬酰胺(Asn)306上;也参见SEQID NO:1。Antibodies without such post-translational modifications in the variable region (e.g. the two variable regions of the heavy chain (both (VH) regions)) are considered "aglycosylated forms" within the scope of the invention, including The variable region of the chain is not glycosylated. However, this "aglycosylated form" may comprise glycosylation in the constant region (C-region) of the antibody, for example, most commonly at a highly conserved glycosylation site in the Fc portion, in particular as defined herein on asparagine (Asn) 306 of the non-variable/constant Fc portion of ; see also SEQ ID NO:1.

本发明方法中的水性蛋白溶液中的蛋白质可以是上文所定义的示例性的“Aβ抗体A”。因此,所述蛋白质可以是Aβ抗体A,其包含如上文所定义的单糖基化Aβ抗体A或双糖基化Aβ抗体A或非糖基化Aβ抗体A,或它们的混合物。The protein in the aqueous protein solution in the method of the present invention may be an exemplary "Aβ antibody A" as defined above. Thus, said protein may be an Aβ antibody A comprising a monoglycosylated Aβ antibody A or a diglycosylated Aβ antibody A or an aglycosylated Aβ antibody A as defined above, or a mixture thereof.

对重组表达的Aβ抗体分子的糖基化亚型的纯化包括下述步骤:Purification of the glycosylated isoforms of recombinantly expressed Aβ antibody molecules comprises the following steps:

(1)蛋白A柱纯化;(1) protein A column purification;

(2)离子交换柱纯化,例如阳离子交换色谱;以及任选地,(2) ion exchange column purification, such as cation exchange chromatography; and optionally,

(3)尺寸排阻柱纯化。(3) Size exclusion column purification.

纯化方案还可包含其它步骤,例如进一步的浓缩步骤(例如渗滤)或分析步骤(例如包括分析柱)。还可采取重复某些特定步骤(例如,可进行两次离子交换色谱步骤)或者省略某些步骤(例如尺寸排阻色谱法)。A purification scheme may also comprise other steps, such as further concentration steps (eg, diafiltration) or analytical steps (eg, including an analytical column). It is also possible to repeat certain steps (eg, two ion exchange chromatography steps may be performed) or to omit certain steps (eg, size exclusion chromatography).

蛋白A是与大多数IgG1的同种型的Fc区域结合的基团特异性配体。其由一些金黄色葡萄球菌菌株合成,并可从中加以分离,将其与色谱粒偶联。也可从商业上获得一些类型的凝胶制备物。可使用的蛋白A柱的例子是MabSelect(商标)柱。理想地,用25mM Tris/HCl、25mM NaCl、5mMEDTA对柱加以平衡,将细胞培养物上清液上样到柱上,用1M Tris/HClpH 7.2洗柱,使用100mM乙酸在pH 3.2时洗脱抗体。Protein A is a group-specific ligand that binds the Fc region of most IgGl isotypes. It is synthesized by some strains of Staphylococcus aureus and can be isolated from which it is coupled to a chromatographic particle. Some types of gel preparations are also commercially available. An example of a protein A column that can be used is a MabSelect (trade mark) column. Ideally, equilibrate the column with 25mM Tris/HCl, 25mM NaCl, 5mM EDTA, load the cell culture supernatant onto the column, wash the column with 1M Tris/HCl pH 7.2, and elute the antibody with 100mM acetic acid at pH 3.2 .

阳离子交换色谱利用了固定相中带正电荷的基团和移动相中样品之间的相互作用。当使用弱阳离子交换剂(例如CM Toyopearl 650

Figure A20088001442000131
)时,进行下述色谱步骤:用100mM乙酸pH 4预平衡、将蛋白A洗脱液上样和用100mM乙酸pH 4洗柱后,通过应用250mM乙酸钠(pH7.8-8.5)和500mM乙酸钠(pH 7.8-8.5)的步骤洗脱抗体和分级分离。采用第一个步骤,通常洗脱出双糖基化亚型流分和单糖基化亚型流分的混合物,使用第二个步骤通常洗脱出非糖基化亚型流分。Cation exchange chromatography exploits the interaction between positively charged groups in the stationary phase and the sample in the mobile phase. When using weak cation exchangers (such as CM Toyopearl 650
Figure A20088001442000131
), the following chromatographic steps were performed: pre-equilibration with 100mM acetic acid pH 4, loading of the Protein A eluate and washing the column with 100mM acetic acid pH 4, followed by Sodium (pH 7.8-8.5) step elution of antibody and fractionation. With the first step, a mixture of fractions of double and monoglycosylated isoforms usually elutes, and with the second step, a fraction of non-glycosylated isoforms usually elutes.

可通过盐步骤从强阳离子交换剂(例如SP Toyopearl 650)洗脱出抗体:用50mM乙酸pH 5.0对柱加以平衡、将pH 4的蛋白A洗脱液上样后,使用50mM乙酸和210mM氯化钠进行第一个洗脱步骤。然后,用50mM乙酸和350mM氯化钠进行第二个洗脱步骤。通过第一个盐步骤,通常洗脱出双糖基化亚型流分和单糖基化亚型流分的混合物,第二个盐步骤通常洗脱出非糖基化亚型。Antibodies can be eluted from strong cation exchangers (e.g. SP Toyopearl 650) by a salt step: equilibrate the column with 50mM acetic acid pH 5.0, load the Protein A eluate at pH 4, and use 50mM acetic acid and 210mM chloride Sodium for the first elution step. Then, a second elution step was performed with 50 mM acetic acid and 350 mM sodium chloride. With the first salt step, a mixture of double and monoglycosylated isoform fractions usually elutes, and the second salt step usually elutes the non-glycosylated isoform.

此外,还可通过盐梯度从强阳离子交换柱(例如SP-Sepharose

Figure A20088001442000141
)洗脱出抗体:在pH4.5下对柱预平衡、上样和洗涤之后,应用从50mM MES pH5.8至50mM MES/1M氯化钠pH 5.8的盐梯度。此时,通常分别洗脱出双糖基化亚型、单糖基化亚型和非糖基化亚型的流分。随后,双糖基化亚型和单糖基化亚型流分可以合并,以得到产品库和/或所需的抗体混合物。In addition, strong cation exchange columns (such as SP-Sepharose
Figure A20088001442000141
) elution of antibodies: After pre-equilibration of the column at pH 4.5, loading and washing, a salt gradient from 50 mM MES pH 5.8 to 50 mM MES/1 M NaCl pH 5.8 was applied. At this point, fractions of the diglycosylated isoform, monoglycosylated isoform and non-glycosylated isoform are usually eluted separately. Fractions of double and monoglycosylated isoforms can then be combined to obtain product libraries and/or desired antibody mixtures.

双糖基化和单糖基化的抗体分子(例如免疫球蛋白)的混合物可通过尺寸排阻色谱法进行进一步纯化。可用的柱的例子是Superdex 200

Figure A20088001442000142
柱。操作缓冲液的例子包括组氨酸/氯化钠,例如10mM组氨酸/125mM氯化钠/pH 6,和磷酸盐缓冲盐水(PBS)。Mixtures of double and monoglycosylated antibody molecules (eg, immunoglobulins) can be further purified by size exclusion chromatography. Examples of available columns are Superdex 200
Figure A20088001442000142
column. Examples of working buffers include histidine/sodium chloride, eg, 10 mM histidine/125 mM sodium chloride/pH 6, and phosphate buffered saline (PBS).

以流穿模式进行阴离子交换色谱、随后进行浓缩/透析是一种备选的纯化步骤。Q Sepharose

Figure A20088001442000143
是阴离子交换步骤的树脂的一个例子。例如,可用37.5mM Tris/HCl pH 7.9对来自SP色谱的洗出液进行三倍稀释,并使其通过预先用25mM Tris/83mM乙酸钠平衡过的Q-Sepharose柱。收集流穿液体,将其调节至pH 5.5,并用例如Hydrosart 30kD
Figure A20088001442000144
膜通过超滤浓缩。随后,浓缩物可在例如10倍体积的20mM组氨酸/HCl pH 5.5中透析。Anion exchange chromatography in flow-through mode followed by concentration/dialysis is an alternative purification step. Q Sepharose
Figure A20088001442000143
is an example of a resin for the anion exchange step. For example, eluate from SP chromatography can be diluted three-fold with 37.5 mM Tris/HCl pH 7.9 and passed through a Q-Sepharose column pre-equilibrated with 25 mM Tris/83 mM sodium acetate. The flow-through is collected, adjusted to pH 5.5, and treated with e.g. Hydrosart 30kD
Figure A20088001442000144
The membrane is concentrated by ultrafiltration. Subsequently, the concentrate can be dialyzed, for example, against 10 volumes of 20 mM histidine/HCl pH 5.5.

如上文所定义的,抗体亚型还可包含处于抗体分子的恒定/非可变部分的其它糖基化,例如在IgG的Fc部分,例如在IgG1的Fc部分中。Fc部分中的所述糖基化涉及高度保守的糖基化,其特征是位于重链的Asn306位置上(例如,根据本文定义的SEQ ID NO:1的重链)。As defined above, antibody subtypes may also comprise other glycosylations in constant/non-variable parts of the antibody molecule, eg in the Fc part of IgG, eg in the Fc part of IgGl. Said glycosylation in the Fc portion involves a highly conserved glycosylation characterized at position Asn306 of the heavy chain (eg heavy chain according to SEQ ID NO: 1 as defined herein).

本发明的制剂中所包含的抗体的IgG-Fc区域可以是同源二聚体,其包含链间二硫键连接的铰链区、糖基化的CH2结构域(其在CH2的天冬酰胺306(Asn-306)处带有N-连接的寡糖)和非共价配对的CH3结构域。Asn-306上糖基化的寡糖是复合双触角(biantennary)型的,可包含核心的庚糖结构,并具有可变添加的外臂糖。The IgG-Fc region of the antibody contained in the formulations of the invention may be a homodimer comprising an interchain disulfide-bonded hinge region, a glycosylated CH2 domain (which is located at asparagine 306 of CH2 (Asn-306) with an N-linked oligosaccharide) and a non-covalently paired CH3 domain. Oligosaccharides glycosylated on Asn-306 are complex biantennary and can contain a core heptose structure with variably added outer arm sugars.

寡糖影响或决定了Fc结构和功能(Jefferis(1998)Immunol Rev.163,50-76)。效应物的功能(编号为具体的特异性的IgG-Fc/效应物配体作用)已被讨论过(Jefferis(2002)Immunol Lett.82(1-2),57-65和Krapp(2003)JMol Biol.325(5),979-89)。该保守的Fc位置Asn306对应于Kabat系统中的“Asn297”(Kabat(1991)Sequences of Proteins of ImmunologicalInterest,第5版,Public Health Service,National Institutes of Health,Bethesda MD.)。Oligosaccharides influence or determine Fc structure and function (Jefferis (1998) Immunol Rev. 163, 50-76). The function of effectors (numbered as specific specific IgG-Fc/effector ligand interactions) has been discussed (Jefferis (2002) Immunol Lett. 82(1-2), 57-65 and Krapp (2003) J Mol Biol. 325(5), 979-89). This conserved Fc position Asn306 corresponds to "Asn297" in the Kabat system (Kabat (1991) Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda MD.).

“稳定剂”这种表述是指能使水性蛋白溶液对抗机械胁迫而化学稳定的可药用稳定剂。此类稳定剂的例子是表面活性剂,例如Tween 20、Tween80、Tween 40、Tween 60、泊洛沙姆、SDS或Triton X或任何其它两亲的分子,或它们的混合物。表面活性剂在水性溶液中的作用已被广泛描述过,它是本领域技术人员公知的。The expression "stabilizer" refers to a pharmaceutically acceptable stabilizer capable of chemically stabilizing an aqueous protein solution against mechanical stress. Examples of such stabilizers are surfactants such as Tween 20, Tween 80, Tween 40, Tween 60, Poloxamer, SDS or Triton X or any other amphiphilic molecule, or mixtures thereof. The effect of surfactants in aqueous solutions has been extensively described and is well known to those skilled in the art.

“注射装置”这种表述指可药用的注射装置。此类装置的一个例子是注射器。The expression "injection device" refers to a pharmaceutically acceptable injection device. An example of such a device is a syringe.

如上文所述,申请人已发现了使水性蛋白溶液对抗机械胁迫而稳定的方法,所述方法包括下述步骤:将所述水性蛋白溶液填装进容器,从而使得所述容器在封闭时基本没有气体顶部空间。As stated above, applicants have discovered a method of stabilizing an aqueous protein solution against mechanical stress, said method comprising the steps of filling said aqueous protein solution into a container such that said container, when closed, is substantially No gas headspace.

在根据本发明的方法的任何实施方式中,可通过例如目测、采用重量或体积方法对没有气体顶部空间加以预先测定。In any embodiment of the method according to the invention, the absence of gas headspace may be predetermined, for example by visual inspection, using gravimetric or volumetric methods.

在根据本发明的方法的任何实施方式中,容器可被填装至超过容器总体积的约97%。In any embodiment of the method according to the invention, the container may be filled to more than about 97% of the total volume of the container.

在根据本发明的方法的任何实施方式中,气体顶部空间可占容器总体积的少于约3%,优选少于2%,更优选少于1%。In any embodiment of the method according to the invention, the gas headspace may comprise less than about 3%, preferably less than 2%, more preferably less than 1% of the total volume of the vessel.

在根据本发明的方法的任何实施方式中,蛋白质可以是抗体,特别是单克隆抗体,例如选自IgG1或IgG2和IgG4,优选地选自针对至少一种下述靶点的有效的单克隆抗体:IGF-1R、CD20、CD19、CCR5、淀粉样蛋白、OX40、EGF受体、VEGF、HER、IL1R、IL13、IL6、IL17或P-选择素。In any embodiment of the method according to the invention, the protein may be an antibody, in particular a monoclonal antibody, for example selected from IgG1 or IgG2 and IgG4, preferably from monoclonal antibodies effective against at least one of the following targets : IGF-1R, CD20, CD19, CCR5, amyloid, OX40, EGF receptor, VEGF, HER, IL1R, IL13, IL6, IL17 or P-selectin.

抗体还可选自本领域已知的和以Actemra、Avastin或Herceptin的名称公开的那些抗体。Antibodies may also be selected from those known in the art and disclosed under the names Actemra, Avastin or Herceptin.

根据本发明的方法还可通过下述事实来表征:水性蛋白溶液中没有用于对抗外源胁迫(特别是机械胁迫)的稳定剂。The method according to the invention can also be characterized by the fact that there are no stabilizers in the aqueous protein solution against exogenous stresses, in particular mechanical stresses.

在根据本发明的方法的任何实施方式中,机械胁迫可以是振荡。机械胁迫通常在约0℃至约40℃之间的温度下发生。In any embodiment of the method according to the invention, the mechanical stress may be oscillation. Mechanical stress typically occurs at a temperature between about 0°C and about 40°C.

在根据本发明的方法中稳定的水性蛋白溶液包含水、蛋白质和其它常规的可药用赋形剂。The stable aqueous protein solution in the method according to the invention comprises water, protein and other conventional pharmaceutically acceptable excipients.

根据本发明的方法可通过下述事实来表征:一旦被稳定,在振荡时水性蛋白溶液基本没有显著的混浊和/或聚集物和/或可见的颗粒。The method according to the invention can be characterized by the fact that, once stabilized, the aqueous protein solution is substantially free of significant turbidity and/or aggregates and/or visible particles upon shaking.

在根据本发明的一种特定实施方式中,容器不是注射装置,例如不是注射器。In a particular embodiment according to the invention, the container is not an injection device, for example not a syringe.

容器可包含与开关装置相连的容纳器部分。在一种实施方式中,容器由与开关装置相连的容纳器组成,优选与打开和密封关闭装置相连的容纳器。The container may comprise a receptacle portion connected to the switching device. In one embodiment, the container consists of a receptacle connected to a switching device, preferably a receptacle connected to an opening and sealing closing device.

本发明还涉及包含与开关装置相连的容纳器部分的容器用于使水性蛋白溶液对抗机械胁迫而稳定的用途。该用途可根据上文所述的本发明方法来实现。The invention also relates to the use of a container comprising a receptacle part connected to a switching device for stabilizing an aqueous protein solution against mechanical stress. This use can be carried out according to the method of the invention as described above.

下述实施例用来阐述本发明,但并非将其限制为本文公开的单独的实施方式。The following examples illustrate the invention but do not limit it to the individual embodiments disclosed herein.

实施例 Example

在下述实施例中,在振荡胁迫下,在两种不同温度、不同填装体积和不同量的聚山梨酯条件下,研究了两种单克隆抗体(IgG1)的聚集行为。使用多种分析技术:目测、浊度、光遮蔽、尺寸排阻色谱法和动态光散射来在尺寸和数量方面对聚集物形成进行检测和监测。In the following example, the aggregation behavior of two monoclonal antibodies (IgG1 ) was studied under shaking stress at two different temperatures, different fill volumes and different amounts of polysorbate. Aggregate formation was detected and monitored in size and number using multiple analytical techniques: visual inspection, turbidity, light obscuration, size exclusion chromatography and dynamic light scattering.

用尺寸为6ml

Figure A20088001442000161
20mm的玻璃瓶来测定容器体积和表面积,它们进而被用于计算每种填装体积的气-液界面程度(通过表面积与体积的比例和顶部空间与体积的比例)。实施例中的所有实验都在这样的瓶中进行。Use size 6ml
Figure A20088001442000161
20 mm glass vials were used to determine the container volume and surface area, which in turn were used to calculate the extent of the gas-liquid interface (by surface area to volume ratio and headspace to volume ratio) for each filled volume. All experiments in the examples were carried out in such bottles.

从下表1和表2可以看出,填装体积越小,顶部空间体积就越大,这是针对与液体样品相互作用的气体体积量的振荡实验过程中的重要方面。填装至最大体积的样品没有顶部空间,振荡期间液体在瓶中的物理运动受到限制。表面积与填装体积的比例随着填装体积的降低而增加,这使振荡过程中玻璃、表面的接触更多(表2)。As can be seen from Tables 1 and 2 below, the smaller the packing volume, the larger the headspace volume, which is an important aspect during an oscillation experiment for the volume of gas interacting with the liquid sample. Samples filled to maximum volume have no headspace and the physical movement of the liquid in the bottle during shaking is limited. The ratio of surface area to packed volume increases with decreasing packed volume, which results in more glass-surface contact during shaking (Table 2).

表1:计算出的6ml

Figure A20088001442000171
20mm瓶的表面积和容器体积Table 1: Calculated 6ml
Figure A20088001442000171
Surface area and container volume of 20mm bottle

表2:计算出的表面积与填装体积和顶部空间与填装体积的比例Table 2: Calculated Surface Area to Packed Volume and Headspace to Packed Volume Ratio

Figure A20088001442000173
Figure A20088001442000173

通过在5℃和25℃,在168小时的时期内选定的时间点,对含有0%、0.0025%、0.005%和0.01%(w/v)的PS20的蛋白制剂随时间进程的情况加以观察,在用橡皮塞和密封铝箔封闭的额定6ml体积的瓶中进行不同填装体积(2.5ml、5.3ml和9ml)的振荡研究。进行该研究是为了评估振荡期间顶部空间对蛋白质稳定性的影响,这是由于蛋白质所暴露的气-液或液-玻璃界面是基于样品瓶中存在的气-液的体积。PS20对蛋白质对抗这些界面以及由此抵抗振荡期间的变性和聚集而加以保护的程度也被评估。图2-9中概括了由于振荡胁迫所致的浑浊、可见的颗粒计数、镜检可见的颗粒和可溶的聚集产物的结果。Protein formulations containing 0%, 0.0025%, 0.005% and 0.01% (w/v) PS20 were observed over time at selected time points over a 168 hour period at 5°C and 25°C , shaking studies of different fill volumes (2.5 ml, 5.3 ml and 9 ml) were performed in bottles of nominal 6 ml volume closed with rubber stoppers and airtight aluminum foil. This study was performed to evaluate the effect of headspace on protein stability during shaking due to the gas-liquid or liquid-glass interface to which the protein is exposed based on the volume of gas-liquid present in the vial. The extent to which PS20 protects the protein against these interfaces and thus against denaturation and aggregation during shaking was also assessed. The results of turbidity due to oscillatory stress, visible particle counts, microscopically visible particles and soluble aggregation products are summarized in Figures 2-9.

从上述结果(图2-9)可以看出,瓶中存在顶部空间对在5℃和25℃下受到振荡胁迫的抗体制剂稳定性都有很大影响。From the above results (Figures 2-9), it can be seen that the presence of headspace in the bottle has a great influence on the stability of antibody formulations subjected to shaking stress at both 5°C and 25°C.

处于“最大”填装体积的所有制剂(图2-5的C栏和图6-7的D栏)以及作为对照的安慰剂(数据未显示)保持稳定,受胁迫的样品表现出与未受胁迫的样品相当的结果。All formulations at the "maximum" fill volume (column C of Figures 2-5 and column D of Figures 6-7), as well as placebo as a control (data not shown), remained stable, with the stressed samples exhibiting similar Stressed samples had comparable results.

填装至最大体积的样品没有显著的顶部空间(少于3%的顶部空间体积),因此液体在瓶内的运动被限制,没有观察到对蛋白的损害。Samples filled to maximum volume had no significant headspace (less than 3% headspace volume), so movement of liquid within the bottle was restricted and no damage to the protein was observed.

与市面上的常规药物产品中所用的一样,具有“标准”的顶部空间时,液体样品在振荡过程中具有在瓶内旋转和泼溅的能力,这导致气-液相互作用,从而使得蛋白不稳定。With a "standard" headspace, as used in conventional pharmaceutical products on the market, the liquid sample has the ability to swirl and splash within the bottle during shaking, which results in gas-liquid interactions so that the protein does not Stablize.

所有样品的液体以同样的方式移动,每种制剂都具有1.1158±0.002mPa.s的动力学粘度。在有和无顶部空间时,振荡时蛋白稳定性之间的差别明显,而在具有体积为2.5ml或5.3ml的顶部空间的样品之间,振荡时没有观察到显著差别。The liquids of all samples moved in the same way and each formulation had a dynamic viscosity of 1.1158 ± 0.002 mPa.s. The difference between protein stability upon shaking was evident with and without headspace, whereas no significant difference was observed upon shaking between samples with headspace volumes of 2.5 ml or 5.3 ml.

在两个温度下针对可见的颗粒计数、浊度和镜检可见的颗粒计数进行分析时,即使少到只存在0.0025%的PS20对空瓶和额定填装量(图2-5,A和B栏;图6-7,B和C栏)中的蛋白质也提供了与不含PS并且填装至最大量的样品(图2-5,C栏;图6-7,D栏)相似的保护。When analyzed for visible particle count, turbidity, and microscopically visible particle count at two temperatures, even as little as 0.0025% PS20 was present for empty bottles and nominal fills (Figure 2-5, A and B columns; Figures 6-7, columns B and C) also provided similar protection to samples containing no PS and packed to the maximum amount (Figures 2-5, column C; Figures 6-7, columns D) .

但是,通过SEC分析,发现对于聚集产物的抑制,尽管0.0025%的PS20在5℃下足以防止Mab A的可溶聚集物形成(图2,最下面一行),但在25℃振荡条件下还需要显著更高的量(0.01%PS20)(图3,最下面一行)。However, by SEC analysis, it was found that for the inhibition of aggregation products, although 0.0025% PS20 was sufficient to prevent the formation of soluble aggregates of Mab A at 5°C (Fig. Significantly higher amounts (0.01% PS20) (Figure 3, bottom row).

因此,振荡胁迫对形成可溶聚集物的负面影响在25℃时要比在5℃时明显得多,尤其是对于有顶部空间(2.5ml和5.3ml)的样品来说。Thus, the negative effect of shaking stress on the formation of soluble aggregates was much more pronounced at 25°C than at 5°C, especially for samples with headspace (2.5ml and 5.3ml).

但是,在整个研究中,在5℃、0%PS20下,具有2.5ml填装体积的样品的浑浊导致比25℃下高得多的FTU值。这种浑浊结果与对Mab A同一样品的镜检可见的颗粒计数和可溶聚集物分析所得的数据并不对应。However, at 5°C, 0% PS20, turbidity of samples with a fill volume of 2.5 ml resulted in much higher FTU values than at 25°C throughout the study. This turbidity result did not correspond to the data obtained from microscopic particle count and soluble aggregate analysis of the same sample of Mab A.

浑浊被描述为:由于各种大小的镜检可见的颗粒散射和吸收了决定液体光学性质的光,导致溶液混浊或模糊。Haze is described as cloudiness or cloudiness of a solution due to microscopically visible particles of various sizes scattering and absorbing light that determine the optical properties of a liquid.

具有5.3ml额定填装体积Mab A和B样品(图3B,6C和7C)显示出随着PS20含量的增加,可溶聚集物和镜检可见的颗粒预期的减少趋势。在2.5ml的Mab A填装体积下也观察到了可溶聚集物形成对PS20浓度的强烈依赖性(图3A,最下面一行),但是有趣的是,与5.3ml填装体积的样品相反,0.0025%PS20的制剂导致了比0%PS20更大量的聚集物。Mab A and B samples with a nominal fill volume of 5.3 ml (Fig. 3B, 6C and 7C) showed the expected trend of reduction of soluble aggregates and microscopically visible particles with increasing PS20 content. A strong dependence of soluble aggregate formation on PS20 concentration was also observed at a Mab A fill volume of 2.5 ml (Fig. 3A, bottom row), but interestingly, in contrast to the 5.3 ml fill volume sample, 0.0025 The formulation of %PS20 resulted in a larger amount of aggregates than 0% PS20.

总之,数据显示,在配制期间填装至“最大量”的振荡样品(即,顶部空间最小化)表现为不需要PS来抑制颗粒或聚集物形成(图2-5,C栏;图6-7,D栏),然而,具有常规顶部空间的样品如果受到胁迫,则需要PS作为稳定剂。所有在玻璃瓶中的市售液体蛋白制剂都有顶部空间(预装填的注射器除外)。目前对于含有蛋白溶液的瓶子所必需的顶部空间体积还没有任何指导或说明。对于商业产品而言,没有顶部空间或仅有最小顶部空间的填装(被定义为封闭的填装体积的97%)对生物医药产品的稳定性有很大价值,使得蛋白溶液中所需的稳定剂最小化。缺少PS20会导致镜检可见的颗粒数量很多,而这一点却通过在同样制剂中不含顶部空间而被完全抑制。Taken together, the data show that shaken samples packed to "maximum volume" (i.e., minimized headspace) during formulation do not appear to require PS to suppress particle or aggregate formation (Fig. 2-5, column C; Fig. 6- 7, column D), however, samples with regular headspace required PS as a stabilizer if they were stressed. All commercially available liquid protein formulations in glass vials have headspace (except prefilled syringes). There is currently no guidance or specification on the necessary headspace volume for bottles containing protein solutions. For commercial products, packing with no or minimal headspace (defined as 97% of the closed packing volume) is of great value to the stability of biopharmaceutical products, allowing the desired Stabilizers are minimized. The absence of PS20 resulted in a high number of microscopically visible particles, which was completely suppressed by the absence of headspace in the same formulation.

另一点是在分析聚集物时正交方法的重要性。如本文中,仅仅基于浊度分析的制剂稳定性表明,0.0025%的PS20足以在25℃振荡期间保护蛋白质,但是与之相反,考虑到SEC数据时必须采用高于0.005%的PS20。Another point is the importance of an orthogonal approach when analyzing aggregates. As herein, formulation stability based solely on turbidity analysis showed that 0.0025% PS20 was sufficient to protect the protein during shaking at 25°C, but in contrast, higher than 0.005% PS20 had to be used considering the SEC data.

数据显示,当受到振荡胁迫时,瓶中顶部空间的存在对蛋白制剂的稳定性有很大影响。The data show that the presence of headspace in the bottle has a strong influence on the stability of the protein formulation when subjected to shaking stress.

填装至最大体积的样品没有显著的顶部空间,因此,在振荡期间液体在瓶中的运动受到限制,没有观察到对蛋白的损害。Samples filled to maximum volume had no significant headspace, therefore, movement of liquid in the vial was restricted during shaking and no damage to the protein was observed.

在不含PS20的制剂中除去顶部空间所产生的在振荡时的保护性能,与存在0.70cm3/cm3和2.61cm3/cm3的顶部空间与体积比例的含有0.0025%PS20的制剂相似,而在0.70cm3/cm3和2.61cm3/cm3这两种体积比例之间没有观察到显著差别。Removal of the headspace in formulations without PS20 resulted in similar protection performance on shaking as formulations containing 0.0025% PS20 in the presence of headspace-to-volume ratios of 0.70 cm 3 /cm 3 and 2.61 cm 3 /cm 3 , No significant difference was observed between the two volume ratios of 0.70 cm 3 /cm 3 and 2.61 cm 3 /cm 3 .

序列表sequence listing

<110>弗·哈夫曼-拉罗切有限公司(F.HOFFMANN-LA ROCHE AG)<110>F.HOFFMANN-LA ROCHE AG

<120>稳定蛋白质的方法<120> Methods of Stabilizing Proteins

<130>24228<130>24228

<140><140>

<141><141>

<150>EP 07107353.0<150>EP 07107353.0

<151>2007-05-02<151>2007-05-02

<160>3<160>3

<170>PatentIn版本3.3<170> PatentIn version 3.3

<210>1<210>1

<211>456<211>456

<212>PRT<212>PRT

<213>人工序列<213> Artificial sequence

<220><220>

<223>人工序列的描述:合成多肽<223> Description of Artificial Sequences: Synthetic Peptides

<400>1<400>1

Gln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

  1               5                  10                  151 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

             20                  25                  3020 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

         35                  40                  4535 40 45

Ser Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser ValSer Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val

     50                  55                  6050 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

 65                  70                  75                  8065 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

                 85                  90                  9585 90 95

Ala Arg Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg TyrAla Arg Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr

            100                 105                 110100 105 110

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala SerPhe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

        115                 120                 125115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser ThrThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

    130                 135                 140130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe ProSer Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145                 150                 155                 160145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly ValGlu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

                165                 170                 175165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu SerHis Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

            180                 185                 190180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr IleSer Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

        195                 200                 205195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys ValCys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

    210                 215                 220210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro AlaGlu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225                 230                 235                 240225 230 235 240

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys ProPro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

                245                 250                 255245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val ValLys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

            260                 265                 270260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

        275                 280                 285275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu GlnAsp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

    290                 295                 300290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His GlnTyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305                 310                 315                 320305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys AlaAsp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

                325                 330                 335325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln ProLeu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

            340                 345                 350340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

        355                 360                 365355 360 365

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro SerLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

    370                 375                 380370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385                 390                 395                 400385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

                405                 410                 415405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val PheSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

            420                 425                 430420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln LysSer Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

        435                 440                 445435 440 445

Ser Leu Ser Leu Ser Pro Gly LysSer Leu Ser Leu Ser Pro Gly Lys

    450                 455450 455

<210>2<210>2

<211>215<211>215

<212>PRT<212>PRT

<213>人工序列<213> Artificial sequence

<220><220>

<223>人工序列的描述:合成多肽<223> Description of Artificial Sequences: Synthetic Peptides

<400>2<400>2

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyAsp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

  1               5                  10                  151 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser SerGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

             20                  25                  3020 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuTyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

         35                  40                  4535 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe SerIle Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

     50                  55                  6050 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu GluGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

 65                  70                  75                  8065 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met ProPro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met Pro

                 85                  90                  9585 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val AlaIle Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

            100                 105                 110100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

        115                 120                 125115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg GluGly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

    130                 135                 140130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerAla Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145                 150                 155                 160145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

                165                 170                 175165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValSer Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

            180                 185                 190180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr LysTyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

        195                 200                 205195 200 205

Ser Phe Asn Arg Gly Glu CysSer Phe Asn Arg Gly Glu Cys

    210                 215210 215

<210>3<210>3

<211>42<211>42

<212>PRT<212>PRT

<213>人(Homo sapiens)<213> Human (Homo sapiens)

<400>3<400>3

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln LysAsp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

  1               5                  10                  151 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile IleLeu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

             20                  25                  3020 25 30

Gly Leu Met Val Gly Gly Val Val Ile AlaGly Leu Met Val Gly Gly Val Val Ile Ala

         35                  4035 40

Claims (28)

1.使水性蛋白溶液对抗外源胁迫而稳定的方法,所述方法包括下述步骤:将所述水性蛋白溶液填装进容器,以使所述容器在封闭时基本没有气体顶部空间。CLAIMS 1. A method of stabilizing an aqueous protein solution against exogenous stress, said method comprising the step of filling said aqueous protein solution into a container such that said container is substantially free of gas headspace when closed. 2.权利要求1所述的方法,其中通过目测、重量或体积方法对没有气体顶部空间加以预先确定。2. The method of claim 1, wherein the absence of gas headspace is predetermined by visual, gravimetric, or volumetric methods. 3.权利要求1或2中任意一项所述的方法,其中所述容器被填装至超过所述容器总体积的约97%。3. The method of any one of claims 1 or 2, wherein the container is filled to greater than about 97% of the total volume of the container. 4.权利要求1至3中任意一项所述的方法,其中所述气体顶部空间占所述容器总体积的少于约3%。4. The method of any one of claims 1 to 3, wherein the gas headspace comprises less than about 3% of the total volume of the vessel. 5.权利要求1至4中任意一项所述的方法,其中所述气体顶部空间占所述容器总体积的少于约2%。5. The method of any one of claims 1 to 4, wherein the gas headspace comprises less than about 2% of the total volume of the vessel. 6.权利要求1至5中任意一项所述的方法,其中所述气体顶部空间占所述容器总体积的少于约1%。6. The method of any one of claims 1 to 5, wherein the gas headspace comprises less than about 1% of the total volume of the vessel. 7.权利要求1至6中任意一项所述的方法,其中所述蛋白是抗体。7. The method of any one of claims 1 to 6, wherein the protein is an antibody. 8.权利要求1至7中任意一项所述的方法,其中所述蛋白是单克隆抗体。8. The method of any one of claims 1 to 7, wherein the protein is a monoclonal antibody. 9.权利要求8所述的方法,其中所述单克隆抗体是IgG1或IgG2或IgG4。9. The method of claim 8, wherein the monoclonal antibody is IgGl or IgG2 or IgG4. 10.权利要求9所述的方法,其中所述单克隆抗体针对至少一种下述靶点:GF-1R、CD20、CD19、CCR5、淀粉样蛋白、OX40、EGF受体、VEGF、HER、IL1R、IL13、IL6、IL17或P-选择素。10. The method of claim 9, wherein the monoclonal antibody is directed against at least one of the following targets: GF-1R, CD20, CD19, CCR5, amyloid, OX40, EGF receptor, VEGF, HER, IL1R , IL13, IL6, IL17 or P-selectin. 11.权利要求10所述的方法,其中所述单克隆抗体选自Actemra、Avastin或Herceptin。11. The method of claim 10, wherein the monoclonal antibody is selected from Actemra, Avastin or Herceptin. 12.权利要求1至11中任意一项所述的方法,其中所述水性蛋白溶液不含对抗外源胁迫的稳定剂。12. The method of any one of claims 1 to 11, wherein the aqueous protein solution is free of stabilizers against exogenous stress. 13.权利要求12所述的方法,其中所述外源胁迫是机械胁迫。13. The method of claim 12, wherein the exogenous stress is mechanical stress. 14.权利要求13所述的方法,其中所述机械胁迫是振荡。14. The method of claim 13, wherein the mechanical stress is oscillation. 15.权利要求1至14中任意一项所述的方法,其中所述外源胁迫发生在0-40℃的温度下。15. The method of any one of claims 1 to 14, wherein the exogenous stress occurs at a temperature of 0-40°C. 16.权利要求1至15中任意一项所述的方法,其中所述水性蛋白溶液包含水和蛋白。16. The method of any one of claims 1 to 15, wherein the aqueous protein solution comprises water and protein. 17.权利要求1至15中任意一项所述的方法,其中所述水性蛋白溶液还包含可药用的赋形剂。17. The method of any one of claims 1 to 15, wherein the aqueous protein solution further comprises a pharmaceutically acceptable excipient. 18.权利要求1至17中任意一项所述的方法,其中所述水性蛋白溶液在振荡时基本没有显著的浑浊和/或聚集物和/或可见的颗粒。18. The method of any one of claims 1 to 17, wherein the aqueous protein solution is substantially free of significant cloudiness and/or aggregates and/or visible particles when shaken. 19.权利要求1至18中任意一项所述的方法,其中所述容器不是注射装置。19. The method of any one of claims 1 to 18, wherein the container is not an injection device. 20.权利要求1至19中任意一项所述的方法,其中所述容器不是注射器。20. The method of any one of claims 1 to 19, wherein the container is not a syringe. 21.权利要求1至20中任意一项所述的方法,其中所述容器包含与开关装置相连的容纳器部分。21. A method as claimed in any one of claims 1 to 20, wherein the container comprises a receptacle portion associated with a switching device. 22.权利要求1至21中任意一项所述的方法,其中所述容器由与开关装置相连的容纳器构成。22. A method as claimed in any one of claims 1 to 21, wherein the container is constituted by a receptacle connected to a switching device. 23.权利要求1至22中任意一项所述的方法,其中所述容器由与打开和密封关闭装置相连的容纳器构成。23. A method as claimed in any one of claims 1 to 22, wherein the container is constituted by a receptacle associated with opening and sealing closure means. 24.包含与开关装置相连的容纳器部分的容器用于使水性蛋白溶液对抗外源胁迫而稳定的用途。24. Use of a container comprising a container portion associated with a switching device for stabilizing an aqueous protein solution against exogenous stress. 25.权利要求24所述的用途,其中所述外源胁迫是机械胁迫。25. The use of claim 24, wherein the exogenous stress is mechanical stress. 26.权利要求25所述的用途,其中所述机械胁迫是振荡。26. The use of claim 25, wherein the mechanical stress is oscillation. 27.权利要求24至26中任意一项所述的用途,其用于权利要求1至25中任意一项所述的方法中。27. The use of any one of claims 24 to 26 in the method of any one of claims 1 to 25. 28.如上所述的本发明。28. The invention as hereinbefore described.
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